[0001] The invention relates to the field of medicine. More particularly the invention relates
to diagnosis. The invention especially relates to quantification of a nucleic acid
of interest in a sample.
[0002] Infections with pathogens are commonly observed all over the world. Such infections
for instance include viral, bacterial, fungal and parasite infections. Early diagnosis
of an infection is often preferred for efficient treatment, which can prevent severe
pathological symptoms. Sometimes, early diagnosis is a prerequisite for a possibility
of treatment, for extending a life-time and/or for improving the quality of life.
Examples of such infections comprise Hepatitis C virus, Hepatitis B virus, tuberculosis,
malaria and HIV, such as HIV-1.
[0003] The HIV-1 epidemic spreads readily over the world and with the spread of the virus
the circulating subtypes of the HIV-1 virus are no longer restricted to one geographical
place on earth. This development requires an application of (nucleic acid) diagnostic
tests that can detect all subtypes of the HIV virus with equal accuracy and precision.
[0004] The globalization of the epidemic and especially the severity in resource-poor countries
(sub-Saharan Africa and south-east Asia) has driven the developed world to assist
the fighting of infection in the less-developed countries with access programs to
pharmaceuticals. However, in order to efficiently help HIV-1 infected people in the
less-developed world there has to be suitable diagnostics in place, for instance to
monitor the efficacy of a treatment. Such suitable diagnostics are especially viral
load assays that measure the amount of HIV-1 RNA in a bodily fluid, such as blood,
blood plasma, mothers milk, semen, lymph fluid, sputum, liquor, saliva and/or urine
of infected individuals. Likewise, suitable nucleic acid load assays are desired for
other infection-related diseases.
[0005] The current status of suitable nucleic acid load assays is of such a high technical
standard that these are not easily transferred to areas located at considerable distance
of technological resources such as in sub-Saharan Africa and/or south-east Asia. This
requires significant investment in infrastructure that is impossible in the setting
of these regions. An alternative solution is an analysis of patient samples in laboratories
in the developed world (for instance Europe and north America). This alternative is
however impossible to exercise due to logistic problems. Body fluid samples such as
blood or blood plasma samples of individuals have to be shipped to the laboratory
in frozen conditions at temperatures well below 0°C, usually in a box with dry ice.
This way of sending clinical material is very expensive and requires the availability
of dry ice at the place of shipment. The latter is often not the case in countries
of the less-developed world. Because of the costs and the unavailability of dry ice
the remote testing of samples in laboratories ("service testing") is not an option
for these countries and for infected people in these countries. The pharmaceutical
access programs for less-developed countries can only be successful if diagnosis is
optimized.
[0006] The invention provides a method for quantifying a nucleic acid of interest in at
least one blood sample, comprising:
- administering said sample to a solid carrier capable of at least in part absorbing
said sample, resulting in at least one spot;
- drying said carrier;
- excising at least one spot from said solid carrier, said at least one spot comprising
at least 250 µl of sample;
- administering said at least one spot to a chaotropic nucleic acid isolation lysis
buffer; so that nucleic acid is extracted from said carrier;
- further purifying the nucleic acid present in said lysis buffer;
- performing a nucleic acid amplification step; and
- quantifying said nucleic acid of interest.
[0007] With a method of the invention a sample, such as a body fluid sample, is stabilized
in such a way that it can be shipped from the site of taking (for instance local hospital
or lab in a less-developed country) and be sent to a service testing laboratory elsewhere
in the world by normal logistics means, for instance normal postal service. Said method
makes possible shipment of a dried sample via surface mail to a remote lab for detection
of a nucleic acid of interest present in a sample.
[0008] A method of the invention can be performed with techniques known in the art. For
instance, a defined amount of liquid sample can be administered to a solid carrier
using a pipette. Said solid carrier is not critical and can comprise any solid carrier
known in the art, as long as it is capable of at least in part absorbing said sample.
For instance, said solid sample can comprise silica. Preferably, however, said solid
carrier comprises filter paper. A filter paper is well capable of absorbing a liquid
sample, while it is of light weight. This is very important for postal services. Said
filter paper can comprise plain untreated filter paper like the 903 paper from Schleicher
and Schuell, or treated filter paper that immobilizes nucleic acid for rapid purification.
The two examples in the field are IsoCode paper from Schleicher and Schuell or FTA-treated
paper from Whatman, which both are bactericidal, fungicidal and virucidal, inhibits
the growth of bacteria and fungi and kills viruses that come in contact with the matrix,
allowing for safe sample handling and sample stability. Another example of a suitable
carrier to store and transport dried fluid samples is a device designed by Lifestock
(
US 5139742) This device contains a small knife with a capillary tube directly attached to it,
which enables collection, storage and transportation of blood that dries in the capillary
tube thereby comparable to the method with the filter paper.
[0009] Said carrier can be dried by different techniques known in the art. Preferably said
sample is dried to the air. This is inexpensive and effective.
[0010] By a representative part of a carrier is meant a part which is indicative for the
amount of sample administered to said solid carrier. For instance said representative
part can comprise the whole of said sample. Said representative part can also comprise
half the amount of said sample. In that case the other half can be used to perform
a second experiment. Said second experiment can be a different experiment, or can
be a similar experiment. In the latter case a certain result can be obtained
in duplo, which is more accurate.
[0011] By a representative amount of nucleic acid is meant an amount which is indicative
for the amount of nucleic acid present in said sample. Said representative amount
can comprise the whole amount of said nucleic acid present in said sample. Alternatively,
said representative amount can comprise a part of said nucleic acid present in said
sample.
[0012] A method of the invention is provided wherein at least 250 µl of sample is administered
to said carrier. More preferably at least 500 µl of sample is administered to said
carrier. With a high sample volume, low titers of nucleic acid of interest can still
be detected. A major drawback in current detection methods is that only nucleic acid
with a concentration above a certain threshold value can be detected. This means that
infected individuals with a low load of pathogenic nucleic acid are not diagnosed
as being infected and, hence, do not get treatment at an appropriate early timepoint.
Until the present invention high amounts of body fluid samples, such as blood or plasma
samples, were not suitable for testing because inhibitory effects were observed. For
instance, it has been reported that hemoglobin and carbonic anhydrase present in whole
blood interfere with the polymerase chain reaction (4). PCR mixtures became deep brown
because of the elution of heme and its degradation products from filter blotters (5).
To improve a PCR amplification, specimens were treated with methanol before the PCR
reaction (6). This involves an extra step, with risk of contaminations and less reliable
test results. Besides, chelating metal ions are reported to act as catalysts for the
breakdown of DNA at a high temperature with low ionic strength (7).
[0013] Surprisingly, with a method of the invention high amounts of sample can be stored
on a dried solid carrier and subsequently analysed. None of the above-mentioned inhibitory
effects are observed and treatment with methanol as a precautionary measure is not
necessary. With a method of the invention it is now possible to analyse large samples
for the presence of a nucleic acid of interest. Hence, low concentrations of a nucleic
acid of interest in a sample can now be detected. A method of the invention is suitable
for screening individuals for the presence of one or more specific pathogens, such
as HIV-1. Alternatively an individual suffering from a disease, or at risk of suffering
from a disease, can be investigated with a method of the invention. Once one or more
foreign nucleic acid(s) are found, it can be determined to which kind of microorganism
it belongs. This can for instance be done by hybridisation protocols using different
kinds of probes. Alternatively, techniques to determine the sequence of the nucleic
acid and analyzing this sequence for homology with any known sequences can be performed.
In one embodiment the invention therefore provides a method of the invention, comprising
identifying said nucleic acid of interest.
[0014] A method of the invention is provided wherein said nucleic acid is quantified. Surprisingly
it is not only possible with a method of the invention to detect a nucleic acid of
interest, but also to determine the
amount of said nucleic acid present in said sample. With a method of the invention no significant
amount of nucleic acid is lost/broken down during storage and/or during the isolation
and/or detection procedure. In the example is shown that, if a method of the invention
is used, a measured amount of a nucleic acid of interest from a dried sample stored
on a solid carrier is comparable to the measured amount of said nucleic acid of interest
when said sample is directly subjected to analysis.
[0015] Dried samples on a solid carrier can now be investigated for the amount of pathogenic
nucleic acid present. Hence, not only the presence, but also the stage of a disease
can now be determined. It is now possible to diagnose individuals of regions with
insufficient facilities, such as inhabitants from resource-poor countries. A body
fluid sample, such as a blood sample, can be collected on a solid carrier such as
a filter paper. Said filter paper can be sent to a laboratory, for instance in Western
Europe. Subsequently the amount of a nucleic acid of interest can be determined and,
hence, the status of a disease. It can also be determined whether a treatment is effective
by determining whether the amount of said nucleic acid declines over time.
[0016] In one aspect the invention provides a method of the invention wherein said solid
carrier is provided with at least two samples. Preferably, said samples are obtained
from the same individual. More preferably, said samples are obtained from the same
body fluid of said individual. Said samples can for instance comprise two aliquots
of blood. Each of said blood samples can be tested independently by a method of the
invention. This way the presence and/or the amount of a nucleic acid of interest in
the blood of said individual can be tested
in duplo. This results in more accurate results. Moreover, each sample can be tested by different
persons/different institutes. Errors made by individuals and/or errors because of
unreliable equipment can be revealed by an independent second measurement.
[0017] In an alternative embodiment, said two samples are used for different purposes. For
instance, one sample can be tested for the amount of viral nucleic acid, while the
other sample can be tested for the amount of bacterial nucleic acid. Both samples
can be used to obtain ratios between viral nucleic acid and chromosomal DNA (as is
important for CMV), or for ratio between mitochondrial vs cellular DNA/RNA or to determine
ratios involving mRNA's or rebosomal RNA's. Many of these tests, however can also
be carried out using only one sample.
[0018] In one embodiment, said solid carrier comprises a series of at least two samples,
taken at different time points. This way a course of a disease can be followed over
time. This embodiment is also particularly suitable for determining whether a treatment
is effective. For instance, a sample can be administered to said solid carrier at
the start of a treatment, and at regular interval afterwards. In this way the said
solid carrier does not only serve the purpose of transportation device, but also for
stable storage at ambient temperatures without degradation of the nucleic acid of
interest. Such solid carrier can be sent to a suitable institute after a certain amount
of time. It can then be established whether a treatment is efficient by quantifying
the amount of pathogenic nucleic acid in each sample by a method of the invention.
It can be established whether said amount declines over time.
[0019] Likewise, samples can be taken from a diseased individual at regular intervals. The
amount of pathogenic nucleic acid in each sample can subsequently be quantified. This
provides more insight into the course of said disease; for instance whether the amount
of pathogenic nucleic acid increases over time, etc.
[0020] It is not necessary to send each sample separately to said institute. A number of
them can be collected over time and be sent at once. This saves time and money. Moreover,
because said samples are sent together, no storing and sorting of separately sent
samples is necessary. A risk of samples getting lost is decreased.
[0021] To even more accurately quantify an amount of a nucleic acid of interest with a method
of the invention, a known amount of a reference nucleic acid can be administered to
said solid carrier. Said reference nucleic acid can be quantified, as well as a nucleic
acid of interest. The accuracy of a quantification of a nucleic acid of interest can
be determined by comparing a measured amount of said reference nucleic acid with the
administered amount of said reference nucleic acid. If said measured amount differs
slightly from the administered amount, the same is likely to be true for said nucleic
acid of interest. Hence, a measured amount of said nucleic acid of interest can be
corrected to obtain an even more accurate result.
[0022] Moreover, with a reference nucleic acid a representative part comprising a part of
a sample can be determined. If a representative part comprising a part of said sample
is provided to said nucleic acid isolation buffer, and the measured amount of reference
nucleic acid appears to be one third of the administered amount, it indicates that
the measured amount of a nucleic acid of interest is also approximately one third
of the amount present in said sample. Hence, a reference nucleic acid shows with which
factor a measured amount of nucleic acid of the invention should be multiplied if
a part of said sample is provided to said nucleic acid isolation buffer. A reference
nucleic acid is preferred which spreads along said solid carrier essentially the same
way as said nucleic acid of interest. In that case it makes no significant difference
which part of said solid carrier is used in a method of the invention. Another way
of accurately quanification of the nucleic acid of interest is by relating it to the
amount of other nucleic acids. In this way it is possible, in either the same reaction
or in separate reactions, in either the same sample or one of the other samples, to
establish a ratio between any DNA and DNA, any RNA and DNA or vice-versa, and any
RNA and RNA target. The invention could comprise applications to determine the ratio
between nucleic acids from the host, e.g. the amount of mitochondrial DNA versus nuclear
DNA as a measure of response to certain HIV therapy, or between nucleic acids from
a pathogen versus those from the host, e.g. the amount of CMV DNA versus host nuclear
DNA. Other applications could comprise the amount of mRNA per cell within the field
of gene expression profiling.
[0023] The invention also provides a method of the invention wherein said representative
part comprises essentially the whole of said at least one sample. It is shown by the
present inventors that even with a large sample a reliable detection and/or quantification
of a nucleic acid of interest is possible with a method of the invention. If the whole
of a large sample is used, even low concentrations of nucleic acid can be detected
and quantified.
[0024] A representative part of said carrier can be provided by cutting a visible spot out
of a solid carrier such as filter paper. This cutting can be done using a normal pair
of scissors, but can also be completely automated, e.g. using equipment from Wallack
to punch out equal surfaces of the solid carrier. Another way of easing this type
of punching by hand, would be to pre-punch said solid carrier, after which the sample
is applied and dried. At arrival in the laboratory for analysis, this pre-punched
part can be easy punched out completely by either specially designed devices or existing
devices as a Safe-lock tube from Eppendorf. Also a representative part of said spot
can be used. However, it is preferred to use the whole carrier, as it is then ensured
that the whole sample is measured. According to the present invention, a solid carrier
such as filter paper does not significantly influence a detection and/or quantification
of a nucleic acid of interest. Therefore, a method of the invention is preferred wherein
said representative part comprises essentially the whole of said solid carrier.
[0025] If in a method of the invention said solid carrier is provided with at least two
samples, said representative part preferably comprises one of said samples. Said representative
part can be used for detecting and/or quantifying a nucleic acid of interest. As has
been explained above, a representative part comprising said second sample can be used
for a measurement
in duplo. Alternatively, an other measurement can be performed.
[0026] A method of the invention is provided wherein said nucleic acid isolation solution
comprises a chaotropic nucleic acid isolation lysis buffer. More preferably, a nucleic
acid isolation buffer as described by Boom et al is used. Preferably said solid carrier
comprises filter paper, since filter paper is cheap, well capable of absorbing a liquid
sample and of light weight which facilitates transport. Typically, elution of the
said nucleic acid takes at least 30 minutes at room-temperature or even shorter at
elevated temperatures, whereas nucleic acid from a bodily fluid that is applied direcly
to the lysis buffer is typically released within 10 minutes. Elevated temperatures
will facilitate efficient and quick elution of the nucleic acid from the solid carrier.
[0027] A method of the invention is particularly suitable for quantifying viral nucleic
acid, especially retroviral nucleic acid. Viral nucleic acid can be present in a latent
stage, which can last for a considerable time. Moreover, a virus such as HIV, HTLV
and HHV is often present during a considerable time within an individual before said
individual experiences any significant symptoms. During that time said virus is often
already transmittable to other persons. Moreover, treatment in an early stage can
improve a chance of recovery, prolong a life-time and/or improve the quality of life.
Therefore it is particularly important to check an individual for the presence of
viral nucleic acid with a method of the invention. Viral nucleic acids to be detected
include sequences from Hepatitis A, B, C, parvovirus, etc. Preferably, said viral
nucleic acid comprises HIV and/or HTLV. More preferably, said viral nucleic acid comprises
HIV-1.
[0028] In one embodiment a method of the invention is provided, wherein said method comprises
genotyping a mutant. This is especially useful for organisms with a fast-changing
genome, such as (retro)viruses. Genotyping is for instance useful for determining
whether a certain treatment is likely to be suitable for an individual patient. Moreover,
if a new mutant is found, an existing pharmaceutical preparation can be adapted, or
a new medicament can be developed.
[0029] A method of the invention is particularly suitable for quantifying a nucleic acid
of interest in a body fluid, such as blood. A sample of such body fluid is easy to
obtain. Obtaining such sample does not cause much inconvenience to an individual,
which would be the case if for instance a biopsy were taken. Moreover, obtaining a
body fluid sample does not require special equipment, which is often lacking in less
developed countries and in remote areas. Preferably a method of the invention is provided
wherein said sample comprises a droplet of whole blood from a finger or heel puncture.
Such finger or heel puncture is commonly taken from newborns, so that said material
is often available without further bothering the individuals. A plasma sample allows
more accurate measurement of an amount of free virus particles, whereas for instance
a blood sample also includes viral nucleic acid integrated within cells.
[0030] To make sure that at least the minimally necessary amount of bodily fluids is collected,
a pre-determined surface can be printed or applied in any other way to the solid carrier.
The meaning is that the surface will be completely filled with the requested bodily
fluid.
[0031] For detection of a nucleic acid of interest often an amplification step (such as
PCR or NASBA) is necessary. Amplified nucleic acid can subsequently be detected using
known methods in the art. A method of the invention therefore comprises an amplification
step. Preferably said amplification comprises real-time monitored amplification. Produced
nucleic acid is directly made visible during such amplification reaction. This can
for instance be achieved by molecular beacon probes or other types of probes. Once
these probes anneal to a template, a fluorescence signal can be generated which can
be monitored during an amplification reaction. The intensity of fluorescence is indicative
for the amount of nucleic acid generated. A calibration curve can be created using
known amounts of nucleic acid. A fluorescence signal from a sample with an unknown
amount of nucleic acid can then be compared with said calibration curve. This way
the amount of said nucleic acid in said sample can be determined, because the intensity
of fluorescence and the amount of nucleic acid are correlated.
[0032] In another embodiment said nucleic acid quantification is performed with an end-point
read-out system. Such systems for instance comprises a colorimetric detection, an
enzymatic assay, and/or a dipstick.
[0033] The invention also provides a use of a dried solid carrier provided with a sample
for detecting, identifying and/or quantifying a nucleic acid of interest in said sample.
As has been described previously, said dried solid carrier can be stored and transported
easily, after which reliable nucleic acid detection and quantification can be carried
out. Preferably said solid carrier comprises at least 250 µl, more preferably at least
500 µl of blood in a dried form. With a use of the invention a large volume of sample
can be investigated, allowing detection and/or quantification of a low concentration
of nucleic acid. The way the solid carrier is designed, it could well contain the
carrier itself that can absorb the bodily fluid, linked to a part of paper or surface
on which information can be written or printed, e.g. information about the patient,
date of sampling or more dates of sampling, therapy regimen, barcodes, ID-numbers,
etc. Such a surface specifically for this type of information is unequivocally linked
t the carrier with sample, thereby making sure the information is not lost. Typically
this type of surfaces can contain much more information than a tube can hold. Logically,
any variation on this theme is possible.
[0034] The invention also uses a kit of parts for detecting, identifying and/or quantifying
a nucleic acid of interest in a sample, comprising:
- a solid carrier capable of at least in part absorbing said sample; and
- a nucleic acid isolation solution.
[0035] Preferably said kit further comprises means for nucleic acid amplification. Said
means for instance comprise means for real-time monitored amplification, and/or means
for amplification with end-point detection/quantification. Such kit of parts is for
instance useful in resource-poor countries with a few hospitals. Such hospitals can
distribute said solid carriers, such as filter papers, among inhabitants in remote
areas. Once samples have been collected, they can be stored and transported to such
hospitals. If said hospital is properly equipped, said samples can be investigated
using said nucleic acid isolation solution. Of course, also hospitals in developed
countries can use such kit of part for collecting and testing a sample. A kit of parts
for detection, identification and/or quantification could well contain a collection
of materials necessary to safely draw the bodily fluid from the patient. Logically
with external bodily fluids like urine, mothers milk or saliva, other safety precautions
will have to be taken than when internal bodily fluids are samples like blood, plasma,
serum, or lymph drain. For the internal bodily fluids, one can compose a kit that
contains a solid carrier capable of at least in part absorbing said sample, and a
nucleic acid isolation solution, next to a pair of examination gloves, a alcohol swab
to clean the skin, a finger or heel puncture device, a bandage, an envelope e.g. with
the address of the destinated laboratory as well as with a space for an identification
number or patient code, and coated inside for safe postal transportation, and a desiccator
to keep the sample dry and to prevent it from fungal or bacterial growth. Other possibilities
for the collection device could comprise of specially designed devices for one-time
use, like a device described in patent number
US5139742: Disposable liquid testing device by Livestock Control Holding B.V. in Amersfoort,
the Netherlands. Any combination of these items, or replaced for other type of items/devices
to be used for storage and/or transportation of any nucleic acid containing bodily
fluids is possible.
[0036] A solid carrier comprising at least the equivalent of 500 µl of blood or a derivative
thereof in dried form is also used. Preferably, said solid carrier comprises at least
two samples. If said samples are of the same kind, said samples can both be tested
separately, resulting in an
in duplo test. Furthermore, a result of a first test can be controlled by independently testing
the other sample. Alternatively, said samples are used for different purposes.
[0037] A solid carrier comprising a series of samples obtained at different data is also
used. As has been described before, such solid carrier is suitable for following a
course of a disease over time, and/or for testing whether a certain treatment is effective.
Preferably a solid carrier comprises a known amount of a reference nucleic acid.
[0038] The invention is further explained in the following example. The example only serves
to clarify the invention. It does not limit the scope of the invention in any way.
Alternative embodiments are also within the scope of the present invention.
Examples
Example 1
[0039] Blood and blood plasma were spotted in 50 µl droplets on S&S 903 paper (Schleicher
& Schull) and dried in the air. Simultaneously, 200 µl of the same blood and plasma
samples were directly added to the lysis buffer as described by Boom et al. (1990).
After drying, the spots on the filter paper were kept at ambient temperature for up
to 3 weeks and can probably be kept at ambient temperature for months. The spots on
the filter paper were excised with a normal pair of scissors and administered to a
tube containing lysis buffer as described by Boom et al. (1990). The filter spots
of 50 µl blood or plasma were added to three different tubes: 1) a 50 ml tube containing
9 ml lysis buffer, 2) a 15 ml tube containing 15 ml lysis buffer or 3) a 1.5 ml eppendorf
tube containing 1 ml lysis buffer.
[0040] The tubes were mildly shaken on a shaking platform for 3 hours at ambient temperature.
During this incubation the blood or plasma spot dissolves from the filter paper into
the lysis buffer. Subsequently the filters were removed from the tubes with a cleaned
pair of tweezers. Between tubes the tweezers were subsequently cleaned with hot water-chlorine-hot
water-70% alcohol.
[0041] To the tubes with lysis buffer and the samples a 1.10
6 copies of a system control RNA molecule were added to allow identification of false
negative reactions at a later stage. The system control RNA is amplified with the
same primers as the wild-type HIV-1 and detected with a distinguishable probe in the
reaction. Due to a length difference the system control RNA can only be amplified
and detected in the absence (or very low amounts) of the wild-type HIV-1 RNA.
[0042] The nucleic acid now present in the lysis buffer was further purified with the method
described by Boom et al (1990) or with dedicated isolation kits purchased from Qiagen
(Qiagen GmbH, Max Volmer Strasse 4, 40724 Hilden, Germany) or Biomerieux (formerly
Organon Teknika, Boseind 15, 5281 RM Boxtel, The Netherlands) and used according to
the manufacturer's protocols. The isolated nucleic acid was stored at -80°C until
further analysis. Usually 5 µl was used as input in NASBA amplification reactions
determining the amount of HIV-1 RNA as described by De Baar et al. (1, 2)
[0043] Standard NASBA nucleic acid amplification reactions were performed in a 20µl reaction
volume and contained: 40mM Tris-pH 8.5, 70mM KCl, 12mM MgCl2, 5mM dithiotreitol, 1mM
dNTP's (each), 2mM rNTP's (each), 0.2µM primer (each), (P1: AAT TCT AAT ACG ACT CAC
TAT AGG GAG AGG GGC GCC ACT GCT AGA GA and P2: CTC AAT AAA GCT TGC CTT GA), 0,05µM
molecular beacon for the wild-type HIV-1 sequence (MB045: FAM-CGA CGT AGT AGT GTG
TGC CCG TCT GTA CGT CG-dabcyl), 0.05µM molecular beacon for the system control RNA
(MB054: ROX-CCG ACT CTC TAC ACA CCA GAC AAA AAA CGA GTC GG-dabcyl) 0.05µM molecular
beacon for the wild-type HIV-1 sequence, 0.05µM molecular beacon for the system control
RNA, 375mM sorbitol, 0.105 µg/µl bovine serum albumin, 6.4 units AMV RT, 32 units
T7 RNA polymerase, 0.08 units RNase H and input nucleic acid. The complete mixture,
except the enzymes was, prior to adding the enzymes, heated to 65°C in order to denature
any secondary structure in the RNA and to allow the primers to anneal. After cooling
the mixture to 41°C the enzymes were added. The amplification took place at 41°C for
90 min in a thermostated fluorimeter (CytoFluor 2000 or EasyQ Reader) and the fluorescent
signal of the molecular beacon probe was measured every minute.
[0044] To achieve quantification, a dilution series of target sequence for a particular
primer set was amplified and the time points at which the reactions became positive
(the time to positivity, TTP) were plotted against the input amounts of nucleic acid.
This way a calibration curve was created that could be used to read TTP values of
reactions with unknown amounts of input and deduce the input amount.
[0045] The results of the determinations in example 1 are shown in table 1 below. By the
results of the system control RNA is appeared that there were no false negative results
and all negative data reported in table 1 are true negative data resulting from the
absence of HIV-1 sequence or presence at concentrations below the detection limit
of the tests.
Patient A |
# 200µl plasma direct to lysis buffer |
200µl blood direct to lysis bufferB |
50 µl plasma spottedB |
200 µl plasma spottedA |
50 µl blood spottedB |
200 µl blood spottedA |
|
|
|
|
|
|
|
R02-05195 |
Neg. |
Pos. |
Neg. |
Neg. |
Neg. |
LQL |
R02-05260 |
Neg. |
Neg. |
Neg. |
Neg. |
Neg. |
Neg. |
R02-05179 |
3.84 |
Pos. |
Pos. |
3.86 |
Pos. |
3.86 |
R02-05183 |
LQL. |
Pos. |
Pos. |
3.74 |
Pos. |
LQL |
R02-05240 |
3.79 |
Pos. |
Pos. |
3.86 |
Pos. |
3.77 |
R02-05244 |
3.79 |
Pos. |
Pos. |
3.59 |
Pos. |
LQL |
R02-05265 |
3.20 |
Pos. |
Neg. |
LQL |
Pos. |
LQL |
R02-05175 |
5.18 |
Pos. |
Pos. |
4.79 |
Pos. |
4.76 |
|
|
|
|
|
|
|
A. The results were determined quantitatively as described in the text with TTP measurements.
The results are given as the Log number. Neg. indicates a negative result, LQL indicates
a positive result, but too low for accurate quantification
B. The determinations of 200 µl blood direct to lysis buffer, 50 µl plasma spotted
and 50 µl blood spotted were not performed quantitatively, only qualitative with either
a positive (Pos.) or negative (Neg.) result. |
[0046] The data in table 1 clearly indicate a good correlation between the results obtained
with the direct admission of sample to the lysis buffer compared to first spotting
of the sample on paper, drying and thereafter admission to the lysis buffer.
Example 2
[0047] Mother milk of 1 woman, spiked with virus from 6 different isolates in 4 concentrations
was spotted in 4 times 50 µl droplets on S&S 903 paper (Schleicher & Schuell), dried
on the air and stored for a minimum of one week at ambient temperature. After drying,
the spots on the filter paper were kept at ambient temperature for up to 3 weeks and
can probably be kept at ambient temperature for months. Simultaneously, 200 µl of
the same mother milk samples was directly added to the lysis buffer as described by
Boom et al. (1990). The spots on the filter paper were excised with a normal pair
of scissors and administered to a tube containing 4 ml lysis buffer as described by
Boom et al. (1990).
[0048] The tubes were mildly shaken on a shaking platform overnight at ambient temperature.
During this incubation the dried spot dissolves from the filter paper into the lysis
buffer. Subsequently the filters were removed from the tubes with a cleaned pair of
tweezers. Between tubes the tweezers were subsequently cleaned with chlorine-hot water-70%
alcohol.
[0049] To the tubes with lysis buffer and sample 1.000.000 copies of a system control RNA
molecule was added to allow identification of false negative reactions at a later
stage. The system control RNA is amplified with the same primers as the wild-type
HIV-1 and detected with a distinguishable probe in the reaction. Due to a length difference
the system control RNA can only be amplified and detected in the absence (or very
low amounts) of the wild-type HIV-1 RNA. The nucleic acid now present in the lysis
buffer was further purified with the method described by Boom et al (1990) or with
dedicated isolation kits purchased from Qiagen (Qiagen GmbH, Max Volmer Strasse 4,
40724 Hilden, Germany) or Biomerieux (formerly Organon Teknika, Boseind 15, 5281 RM
Boxtel, The Netherlands) and used according to the manufacturer's protocols. The isolated
nucleic acid was stored at -80°C until further analysis. Usually 5 µl was used as
input in NASBA amplification reactions determining the amount of HIV-1 RNA as described
by De Baar et al.
[0050] Standard NASBA nucleic acid amplification reactions were performed in a 20µl reaction
volume and contained: 40mM Tris-pH 8.5, 70mM KCl, 12mM MgCl2, 5mM dithiotreitol, 1mM
dNTP's (each), 2mM rNTP's (each), 0.2µM primer (each) (P1: AAT TCT AAT ACG ACT CAC
TAT AGG GAG AGG GGC GCC ACT GCT AGA GA and P2: CTC AAT AAA GCT TGC CTT GA), 0.05µM
molecular beacon for the wild-type HIV-1 sequence (MB045: FAM-CGA CGT AGT AGT GTG
TGC CCG TCT GTA CGT CG-dabcyl), 0.05µM molecular beacon for the system control RNA
(MB054: ROX-CCG ACT CTC TAC ACA CCA GAC AAA AAA CGA GTC GG-dabcyl), 375mM sorbitol,
0.105 µg/µl bovine serum albumin, 6.4 units AMV RT, 32 units T7 RNA polymerase, 0.08
units RNase H and input nucleic acid. The complete mixture, except the enzymes was,
prior to adding the enzymes, heated to 65°C in order to denature any secondary structure
in the RNA and to allow the primers to anneal. After cooling the mixture to 41°C the
enzymes were added. The amplification took place at 41°C for 60 min in a thermostated
fluorimeter (CytoFluor 2000 or EasyQ Reader) and the fluorescent signal of the molecular
beacon probe was measured every minute.
[0051] To achieve quantification, a dilution series of target sequence for a particular
primer set was amplified and the time points at which the reactions became positive
(the time to positivity, TTP) were plotted against the input amounts of nucleic acid.
This way a calibration curve was created that could be used to read TTP values of
reactions with unknown amounts of input and deduce the input amount.
[0052] The results of the determinations of the same samples spotted and added directly
to the lysis buffer were compared and the analysis is shown in figure 1 below. By
the results of the system control RNA it appeared that there were no false negative
results and all negative data reported in figure 1 are true negative data resulting
from the absence of HIV-1 sequence or presence at concentrations below the detection
limit of the tests.
Example 3
[0053] Plasma of 88 HIV-1 infected individuals was spotted in 200 µl droplets on S&S 903
paper (Schleiger & Schull) and dried in the air and stored for a minimum of 24 hours
at ambient temperature. Simultaneously, 200 µl of the same plasma samples was directly
added to the lysis buffer as described by Boom et al. (1990). The spots on the filter
paper were pinched out and administered to a tube containing 4 ml lysis buffer as
described by Boom et al. (1990).
[0054] The tubes were mildly shaken on a shaking platform for 3 hours at ambient temperature.
During this incubation the dried spot dissolves from the filter paper into the lysis
buffer. Subsequently the filters were removed from the tubes with a cleaned pair of
tweezers. Between tubes the tweezers were subsequently cleaned with hot water-chlorine-hot
water-70% alcohol.
[0055] The nucleic acid now present in the lysis buffer was further purified with the method
described by Boom et al (1990) or with dedicated isolation kits purchased from Qiagen
(Qiagen GmbH, Max Volmer Strasse 4, 40724 Hilden, Germany) or Biomerieux (formerly
Organon Teknika, Boseind 15, 5281 RM Boxtel, The Netherlands) and used according to
the manufacturer's protocols. The isolated nucleic acid was stored at -80°C until
further analysis. Usually 5 µl was used as input in NASBA amplification reactions
determining the amount of HIV-1 RNA as described by De Baar et al [2,3].
[0056] Standard NASBA nucleic acid amplification reactions were performed in a 20µl reaction
volume and contained: 40mM Tris-pH 8.5, 70mM KCl, 12mM MgCl2, 5mM dithiotreitol, 1mM
dNTP's (each), 2mM rNTP's (each), 0.2µM primer (each), 0.05µM molecular beacon for
the wild-type HIV-1 sequence, 0.05µM molecular beacon for the system control RNA,
375mM sorbitol, 0.105 µg/µl bovine serum albumin, 6.4 units AMV RT, 32 units T7 RNA
polymerase, 0.08 units RNase H and input nucleic acid. The complete mixture, except
the enzymes was, prior to adding the enzymes, heated to 65°C in order to denature
any secondary structure in the RNA and to allow the primers to anneal. After cooling
the mixture to 41°C the enzymes were added. The amplification took place at 41°C for
90 min in a thermostated fluorimeter (CytoFluor 2000 or EasyQ Reader) and the fluorescent
signal of the molecular beacon probe was measured every minute.
[0057] To achieve quantification, a dilution series of target sequence for a particular
primer set was amplified and the time points at which the reactions became positive
(the time to positivity, TTP) were plotted against the input amounts of nucleic acid.
This way a calibration curve was created that could be used to read TTP values of
reactions with unknown amounts of input and deduce the input amount.
[0058] The results of the determinations of the same samples spotted and added directly
to the lysis buffer were compared and the analysis is shown in figure 2.
[0059] The data in figure 2 clearly indicate a very good correlation between the results
obtained with the direct admission of sample to the lysis buffer compared to first
spotting of the sample on paper, drying and thereafter admission to the lysis buffer.
When analyzed with the Pearson correlation test a correlation coefficient (r) of 0.919
for plasma direct and 0.959 for dried plasma was found.
Figure legends
[0060]
Figure 1. Comparison of quantitative HIV-1 data obtained on mother milk samples that were analyzed
directly or were first spotted and dried on filter paper. The assay cut of is at log2,
indicated by solid lines in the graph. The numbers on the axis indicates the log copy
number of HIV-1 RNA molecules found in the test.
The data in figure 1 clearly indicate a very good correlation between the results
obtained with the direct admission of sample to the lysis buffer compared to first
spotting of the sample on paper, drying and thereafter admission to the lysis buffer.
Figure 2. Comparison of quantitative HIV-1 data obtained on plasma samples that were analyzed
directly or were first spotted and dried on filter paper. The assay lower limit of
detection is at log2, indicated by solid lines in the graph. The numbers on the axis
indicates the log copy number of HIV-1 RNA molecules found in the test.
References
[0061]
- 1. Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noordaa J,
1990. Rapid and simple method for purification of nucleic acids. J Clin Microbiol;
28(3):495-503.
- 2. de Baar MP, van Dooren MW, de Rooij E, Bakker M, van Gemen B, Goudsmit J, de Ronde A.
Single rapid real-time monitored isothermal RNA amplification assay for quantification
of human immunodeficiency virus type 1 isolates from groups M, N, and O. J Clin Microbiol.
2001 Apr;39(4):1378-84.
- 3. de Baar MP, Timmermans EC, Bakker M, de Rooij E, van Gemen B, Goudsmit J. One-tube real-time
isothermal amplification assay to identify and distinguish human immunodeficiency
virus type 1 subtypes A, B, and C and circulating recombinant forms AE and AG. J Clin
Microbiol. 2001 May;39(5):1895-902.
- 4. Caggana M, Conroy J, Pass K. Rapid, efficient method for multiplex amplification from
filter paper. Human mutation 1998; 11: 404-409.
- 5. Romppanen E, Mononen I. PCR-oligonucleotide ligation assay from dried blood spots.
Clinical Chemistry 1999; 45(11): 2022-2025.
- 6. Romppanen E. Oligonucleotide ligation assay: applications to molecular diagnosis
of inherited disorders. Scand J Clin Lab Invest 2001; 61: 123-130.
- 7. Tzeng CC, Lin SJ, Chen YJ, Kuo PL, Jong YJ, Tsai LP, Chen RM. An effective strategy
of using molecular testing to screen mentally retarded individuals for fragile X syndrome.
Diagnostic Molecular Pathology 2001; 10: 34-40.
1. A method for quantifying a nucleic acid of interest in at least one blood sample,
comprising:
- administering said sample to a solid carrier capable of at least in part absorbing
said sample, resulting in at least one spot;
- drying said carrier;
- excising at least one spot from said solid carrier, said at least one spot comprising
at least 250 µl of sample;
- administering said at least one spot to a chaotropic nucleic acid isolation lysis
buffer; so that nucleic acid is extracted from said carrier;
- further purifying the nucleic acid present in said lysis buffer;
- performing a nucleic acid amplification step; and
- quantifying said nucleic acid of interest.
2. A method according to claim 1 comprising identifying said nucleic acid.
3. A method according to any one of claims 1-2, wherein said solid carrier is provided
with at least two samples.
4. A method according to any one of claims 1-3, comprising administering to said solid
carrier a known amount of a reference nucleic acid.
5. A method according to any one of claims 1-4, wherein said part comprises the whole
of said at least one sample.
6. A method according to claim 3 or 4, wherein said part comprises one of said samples.
7. A method according to any one of claims 1-6, wherein said nucleic acid comprises RNA.
8. A method according to claim 7 wherein said RNA comprises mitochondrial RNA, viral
RNA and/or messenger RNA.
9. A method according to any one of claims 1-8, wherein viral nucleic acid is detected.
10. A method according to claim 9, wherein said viral nucleic acid comprises a retroviral
nucleic acid.
11. A method according to claim 9 or 10, wherein said viral nucleic acid comprises HIV
and/or HTLV.
12. A method according to any one of claims 9-11, wherein said viral nucleic acid comprises
HIV-1.
13. A method according to any one of claims 1-12, wherein said carrier comprises a filter-paper.
14. A method according to any one of claim 1-13, comprising genotyping a mutant.
15. A method according to any one of claims 1-14, wherein said sample comprises a droplet
of whole blood from a finger or heel puncture.
16. A method according to claim 15, wherein said amplification comprises real-time monitored
amplification.
17. A method according to any one of claims 1-16, wherein said nucleic acid detection
and/or quantification is performed with an end-point read-out system.
18. A method according to any one of claims 1-17 wherein a ratio between different nucleic
acids is determined.
1. Verfahren zur Quantifizierung einer Nucleinsäure von Interesse in wenigstens einer
Blutprobe, umfassend:
- Zufügen der Probe zu einem festen Träger, der fähig ist, wenigstens teilweise die
Probe zu absorbieren, was in wenigstens einem Fleck resultiert;
- Trocknen des Trägers;
- Ausschneiden wenigstens eines Flecks aus dem festen Träger, wobei wenigstens ein
Fleck wenigstens 250 µl Probe umfasst;
- Zufügen des wenigstens einen Flecks zu einem chaotropen Nucleinsäure-Isolierungslysepuffer,
so dass die Nucleinsäure aus dem Träger extrahiert wird;
- weitere Reinigung der Nucleinsäure, die in dem Lysepuffer vorliegt;
- Durchführen eines Nucleinsäure-Amplifikations-Schritts; und
- Quantifizieren der Nucleinsäure von Interesse.
2. Verfahren gemäß Anspruch 1, das ein Identifizieren der Nucleinsäure umfasst.
3. Verfahren gemäß einem der Ansprüche 1 bis 2, wobei der feste Träger mit wenigstens
zwei Proben versehen wird.
4. Verfahren gemäß einem der Ansprüche 1 bis 3, umfassend Zugeben einer bekannten Menge
einer Referenznucleinsäure zu dem festen Träger.
5. Verfahren gemäß einem der Ansprüche 1 bis 4, wobei der Teil die Gesamte der wenigstens
einen Probe umfasst.
6. Verfahren gemäß Anspruch 3 oder 4, wobei der Teil eine der Proben umfasst.
7. Verfahren gemäß einem der Ansprüche 1 bis 6, wobei die Nucleinsäure RNA umfasst.
8. Verfahren gemäß Anspruch 7, wobei die RNA mitochondriale RNA, virale RNA und/oder
Messenger-RNA umfasst.
9. Verfahren gemäß einem der Ansprüche 1 bis 8, wobei virale Nucleinsäure detektiert
wird.
10. Verfahren gemäß Anspruch 9, wobei die virale Nucleinsäure eine retrovirale Nucleinsäure
umfasst.
11. Verfahren gemäß Anspruch 9 oder 10, wobei die virale Nucleinsäure HIV und/oder HTLV
umfasst.
12. Verfahren gemäß einem der Ansprüche 9 bis 11, wobei die virale Nucleinsäure HIV-1
umfasst.
13. Verfahren gemäß einem der Ansprüche 1 bis 12, wobei der Träger ein Filterpapier umfasst.
14. Verfahren gemäß einem der Ansprüche 1 bis 13, das ein Genotypisieren einer Mutante
umfasst.
15. Verfahren gemäß einem der Ansprüche 1 bis 14, wobei die Probe einen Tropfen Vollblut
aus einer Finger- oder Fersenpunktion umfasst.
16. Verfahren gemäß Anspruch 15, wobei die Amplifikation eine Echtzeit-überwachte Amplifikation
umfasst.
17. Verfahren gemäß einem der Ansprüche 1 bis 16, wobei die Nucleinsäuredetektion und/oder
-quantifizierung mit einem Endpunkt-Ablese-System durchgeführt wird.
18. Verfahren gemäß einem der Ansprüche 1 bis 17, wobei ein Verhältnis zwischen verschiedenen
Nucleinsäuren bestimmt wird.
1. Un procédé pour quantifier un acide nucléique d'intérêt dans, au moins, un échantillon
sanguin comprenant :
- l'administration dudit échantillon à un support solide capable d'absorber au moins
en partie ledit échantillon, de façon à obtenir au moins une tache;
- le séchage dudit support;
- la découpe d'au moins une tache dudit support solide, ladite au moins une tache
comprenant au moins 250µl d'échantillon;
- l'administration de ladite au moins une tache sur un tampon de lyse chaotropique
pour l'isolation de l'acide nucléique de sorte que l'acide nucléique soit extrait
dudit support;
- la purification additionnelle de l'acide nucléique présent dans ledit tampon de
lyse;
- la réalisation d'une étape d'amplification d'un acide nucléique; et
- la quantification dudit acide nucléique d'intérêt.
2. Un procédé selon la revendication 1 comprenant l'identification dudit acide nucléique.
3. Un procédé selon une quelconque des revendications 1-2, dans lequel ledit support
solide comporte au moins deux échantillons.
4. Un procédé selon une quelconque des revendications 1-3, comprenant l'administration
audit support solide d'une quantité connue d'un acide nucléique de référence.
5. Un procédé selon une quelconque des revendications 1-4, dans lequel ladite partie
comprend l'ensemble dudit au moins un échantillon.
6. Un procédé selon l'une des revendications 3 ou 4, dans lequel ladite partie comprend
un desdits échantillons.
7. Un procédé selon une quelconque des revendications 1-6, dans lequel ledit acide nucléique
comprend de l'ARN.
8. Un procédé selon la revendication 7, dans lequel ledit ARN comprend un ARN mitochondrial,
un ARN viral et/ou un ARN messager.
9. Un procédé selon une quelconque des revendications 1-8, dans lequel est détecté de
l'acide nucléique viral.
10. Un procédé selon la revendication 9, dans lequel ledit acide nucléique viral comprend
un acide nucléique rétroviral.
11. Un procédé selon la revendication 9 ou 10, dans lequel ledit acide nucléique viral
comprend le VIH et/ou le HTLV.
12. Un procédé selon une quelconque des revendications 9-11, dans lequel l'acide nucléique
viral comprend le VIH-1.
13. Un procédé selon une quelconque des revendications 1-12, dans lequel ledit support
comprend un filtre en papier.
14. Un procédé selon une quelconque des revendications 1-13, comprenant le génotypage
d'un mutant.
15. Un procédé selon une quelconque des revendications 1-14, dans lequel ledit échantillon
comprend une gouttelette de sang total prélevée sur un doigt ou ponctionnée au talon.
16. Un procédé selon la revendication 15, dans lequel ladite amplification comprend une
amplification controlée en temps réel.
17. Un procédé selon une quelconque des revendications 1-16, dans lequel la détection
de l'acide nucléique et/ou sa quantification est réalisée avec un système de lecture
du point de virage.
18. Un procédé selon une quelconque des revendications 1-17, dans lequel est déterminé
un ratio entre différents acides nucléiques.